The field of the invention relates to mutations to produce a fungal glucoamylase enzyme that is more selective for the production of glucose rather than the xcex1-1,6 linked disaccharide isomaltose, is more thermostable, and has increased pH optimum and produces increased amounts of glucose compared to wildtype enzymes.
Glucoamylase (EC 3.2.1.3) is a carbohydrase. Discovered in 1951, it is an exo-hydrolase that cleaves D-glucose from the nonreducing ends of maltooligosaccharides, attacking xcex1-(1,4)-, and at a much slower rate, xcex1-(1,6)-glucosidic bonds. It is one of more than one hundred carbohydrases (EC 3.2.1) that cleave O-glycosidic bonds of either xcex1- or xcex2-configuration. The functional and structural relatedness of these enzymes is reflected in the presence of at least three discrete regions of sequence homology between glucoamylase and several xcex1-amylases, xcex1-glucosidases, and transglucanosylases [Svensson, 1988], and a similar domain structure to carbohydrases that attack insoluble substrates [Knowles et al., 1987; Svensson et al., 1989)]. Aspergillus awamori glucoamylase (1,4-xcex1-D-glucan glucohydrolase; EC 3.2.1.3) is one of the most important of the glucoamylases.
Glucoamylase is primarily used in industry for the production of high-fructose corn sweeteners in a process that involves 1) xcex1-amylase to hydrolyze starch to maltooligosaccharides of moderate length (dextrin); 2) Glucoamylase to hydrolyze dextrin to glucose; and 3) glucose isomerase to convert glucose to fructose. Corn sweeteners have captured over 50% of the U. S. sweetener market, and the three enzymes used to make them are among the enzymes made in highest volume. In addition, glucose produced by glucoamylase can be crystallized or used in fermentation to produce organic products such as citric acid, ascorbic acid, lysine, glutamic acid or ethanol for beverages and fuel. Approximately 12% of the country""s corn production is processed with glucoamylase. Although glucoamylase has been successfully used for many years, it would be a more attractive product if it produced higher amounts of glucose instead of disaccharides, if it were more stable, and if it could be used in the same vessel with glucose isomerase.
Glucoamylase does not give 100% yield of glucose from dextrin because it makes various di- and trisaccharides, especially isomaltose and isomaltotriose, from glucose [Nikolov et al., 1989]. These products, formed at high substrate concentrations, result from the ability of glucoamylase to form xcex1-(1,6)-glucosidic bonds. Glucoamylase is not as thermostable as either xcex1-amylase or glucose isomerase. The optimum pH of GA (pH4-4.5) is lower than that of xcex1amylase (pH5.5-6.5) and glucose isomerase (pH7-8). Therefore glucoamylase hydrolysis must be done separately from the other enzymatic reactions in a different vessel and at lower temperatures, causing higher capital costs.
Glucoamylase from the filamentous fungus Aspergillus niger is the most widely used glucoamylase, and its bio-chemical properties have been extensively characterized. This enzyme is found mainly in two forms, GAI (616 amino acids; referred to as AA hereinafter) and GAII (512 AA), differing by the presence in GAI of a 104-AA C-terminal domain required for adsorption to native starch granules [Svensson et al., 1982; Svensson et al., 1989]. Both forms have a catalytic domain (AA1-440) followed by a Ser/Thr-rich, highly O-glycosylated region (AA441-512) [Gunnarsson et al., 1984]. The first thirty residues of this region are included in the three-dimensional structure of the enzyme [Aleshin et al., 1994; 1996; Stoffer et al., 1995]; they wrap around the catalytic domain like a belt. There is strong AA sequence homology among fungal glucoamylase""s in four distinct regions of the catalytic domain that correspond to the loops that form the substrate binding site [Itoh et al., 1987]. In A. niger glucoamylase these regions are AA35-59, AA104-134, AA162-196, and AA300-320. The second and third regions partially or completely overlap the three regions of homology to xcex1-amylases [Svensson, 1988]. In addition, the raw starch binding domain (AA512-616) has high homology to similar domains from several starch-degrading enzymes [Svensson et al., 1989].
Kinetic analysis showed that the substrate binding site is composed of up to seven subsites [Savel""ev et al., 1982] with hydrolysis occurring between subsites 1 and 2. The pKa""s of hydrolysis, 2.75 and 5.55 [Savel""ev and Firsov, 1982], suggest that carboxylic acid residues at subsites 1 and 2 provide the catalytic acid and base for hydrolysis. Chemical modification experiments showed that three highly conserved residues, Asp176, Glu179, and Glu180, are protected and are in the active site, suggesting that one or more of them are the possible catalytic residues [Svensson et al., 1990]. Chemical modification experiments also indicated that the highly conserved residue Trp120 is essential, and is located in subsite 4 [Clarke and Svensson, 1984]. Trp120 is homologous to Trp83 of Aspergillus oyzae xcex1-amylase [Clarke and Svensson, 1984], which is also located in the active site of that enzyme [Matsuura et al., 1984]. Site directed mutagenesis studies have indicated that Glu179 is the catalytic acid residue, while Glu400 is the catalytic base residue [Frandse et al, 1994; Harris et al, 1993; Sierks et al, 1990]
Glucoamylases from A. niger [Svensson et al., 1983; Boel et al., 1984] and Aspergillus awamori [Nunberg et al., 1984] have been cloned and sequenced, and have identical primary structures. Innis et al. [1985] and more recently Cole et al. [1988] have developed vectors (pGAC9 and pPM18, respectively) for glucoamylase expression in yeast, allowing convenient manipulation and testing of glucoamylase mutants.
According to the present invention, a fungal glucoamylase (1,4-xcex1-D-glucan glucohydrolase; EC 3.2.1) with decreased thermal inactivation (increased thermostability) and reduced isomaltose formation provided by the mutation Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two is provided. Cumulative thermostability is also provided for GA by including the mutation Asn20Cys coupled with Ala27Cys and at least one mutation from Table 13. An engineered GA including Ser30Pro, Gly137Ala, and Asn20Cys coupled with Ala27Cys provides even more thermostability. Cumulative thermostability is also provided for GA by including the mutation Asn20Cys coupled with Ala27Cys and at least two mutations from Table 13.
The present invention also provides a fungal glucoamylase with reduced isomaltose formation including an Asn20Cys coupled with Ala27Cys mutation (S-S mutation) and at least one mutation selected from Table 14. In an embodiment Asn20Cys coupled with Ala27Cys mutation and a 311-314Loop (also referred to as 300Loop) mutation are included in an engineered GA. In a further preferred embodiment the engineered glucoamylase with reduced isomaltose formation includes Asn20Cys coupled with Ala27Cys mutations Ser30Pro and Gly137Ala.
The present invention also provides engineered fungal glucoamylase including a 311-314Loop mutation whereby reduced isomaltose formation is provided by the mutation.
In a further embodiment fungal glucoamylase including a 311-314Loop mutation and at least one mutation from Table 14 are prepared whereby cumulative reduced isomaltose formation is provided by the additional mutation.
The present invention provides a fungal glucoamylase including a mutation Ser411Ala whereby increased pH optimum and reduced isomaltose formation is provided by the mutation. In an embodiment the Ser411Ala mutation is combined with at least one mutation from Table 15 whereby cumulative increased pH optimum is provided by the mutations. In an embodiment the Ser411Ala mutation is combined with at least one mutation from Table 14 whereby cumulative reduced isomaltose formation is provided by the mutations.
In a further embodiment an engineered fungal glucoamylase includes a mutation Ser411Ala and a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair whereby increased thermal stability, increased pH optimum and reduced isomaltose formation are provided by the mutations.
In a still further embodiment a fungal glucoamylase is engineered to include a Ser411Ala mutation and a mutation pair Asn20Cys coupled with Ala27Cys forming a disulfide bond between the two members of the pair and a 311-314Loop mutation whereby increased thermal stability, increased pH optimum and reduced isomaltose formation are provided by the mutations.
The present invention provides a method to obtain a fungal glucoamylase with reduced isomaltose formation by designing mutations to decrease the xcex1-(1,6)-glucosidic linkage affinity of GA.
The present invention also provides a method to obtain a fungal glucoamylase with decreased thermal inactivation by designing mutations to decrease the enzyme""s conformational entropy of unfolding and/or increase stability of xcex1-helices, increase disulfide bonds, hydrogen bonding, electrostatic interactions, hydrophic interactions, Vanderwalls interactions and packing compactness.
The present invention also provides a fungal glucoamylase with increased pH optimum including changing the polarity, charge distribution and hydrogen bonding in the microenvironment of the catalytic base Glu400.
The present invention also provides a method of genetically engineering glucoamylase carrying at least two cumulatively additive mutations. Individual mutations are generated by site-directed mutagenesis. These individual mutations are screened and those selected which show increased pH optimum and which show decreased irreversible thermal inactivation rates or reduced isomaltose formation. Site directed mutagenesis is then performed to produce enzymes carrying at least two of the isolated selected mutations. Finally the engineered enzymes are screened for cumulatively additive effects of the mutations on thermal stabilizing or reduced isomaltose formation by the produced enzymes carrying at least two of the isolated selected mutations. Alternatively, the engineered enzyme is screened for cumulatively additive effects of both of the mutations on pH optimum, thermostability and/or reduced isomaltose formation by the produced enzymes carrying at least two of the isolated selected mutations.
Vectors for each of the mutations and mutation combinations are also provided by the present invention as well as host cells transformed by the vectors. Also provided is a fungal glucoamylase including a mutation of Ser30Pro coupled with at least two stabilizng mutations forming a disulfide bond between the two stabilizing members. A fungal glucoamylase including a Ser30Pro/Gly137Ala/311-314 Loop is provided. A fungal glucoamylase including a mutation Ser30Pro/Glu137Ala/Ser411Ala is also provided.